This invention relates to a chemical modification of biologically active proteins which alters the chemical and/or physiological properties of these proteins. More specifically, this invention relates to selective conjugation of lipophilic poorly watersoluble proteins to heparin fragments to render the proteins more soluble at physiological pH.
Many heterologous proteins produced in microbial host cells are found as poorly soluble or insoluble material in refractile bodies. Examples of heterologous proteins which form refractile bodies in commonly found culture conditions include interleukin-2 (IL-2), interferon-.beta. (IFN-.beta.), feline leukemia virus (FeLV) envelope protein, human growth hormone (hGH), bovine growth hormone (bGH), porcine growth hormone (pGH), and certain proteins coated or fused with a virus such as FMD virus. Many of these proteins are hydrophobic in nature and tend to stick to materials and to themselves (i.e., aggregate) rather than remain in solution. Also, many of these recombinant proteins are unglycosylated, whereas their native counterparts are glycosylated water-soluble molecules. Modifications of these proteins which might alter their solubility properties would be desirable to increase production yields as well as to facilitate their formulation for therapeutic use. In addition, modifications may reduce or eliminate aggregation of the protein when it is introduced in vivo, thereby reducing its immunogenicity.
The use of polypeptides for the purpose of producing a particular physiological response is well known in the medicinal arts. A limitation to the potential therapeutic benefit derived from the clinical use of polypeptides is their, ability to elicit an immune response. This immune response may be caused or aggravated by aggregates in the material prior to injection as described by R. Illig (1970), J. Clin. Endrocr., 31, 679-688, W. Moore (1978), J. Clin. Endrocrinol. Metab., 46, 20-27 and W. Moore and P. Leppert (1980), J. Clin. Endrocrinol. Metab., 51, 691-697. This response involves the production of antibodies to the polypeptides. This antibody production may decrease or eliminate the desired biological function of the polypeptide, sometimes by causing reduced residence time in the circulatory system (reduced half-life) or by modifying the molecule by virtue of the antibody-polypeptide interaction.
Modification of these potentially useful therapeutic polypeptides so as to preclude or at least reduce an immune response while still maintaining desired physiological a ctivities of the polypeptide would allow the use of these polypeptides in the mammalian circulatory system without the aformentioned disadvantages. In addition, due to the increased half-life of the circulating polypeptide, smaller amounts of the polypeptide would be required for the desired therapeutic effect than have heretofore been possible.
The problems of immunogenicity and short half-life in circulation set forth hereinabove and other undesirable properties of certain proteins are well recognized and various modifications of polypeptides have been undertaken to solve them. These include the modification of proteins with substantially straight chain polymers such as polyethylene glycol (PEG).
For example, U.S. Pat. No. 4,261,973 describes conjugation of immunogenic allergen molecules with non-immunogenic water-soluble polymers such as PEG to reduce the immunogenicity of the allergen.
U.S. Pat. No. 4,055,635 discloses pharmaceutical compositions comprising a water-soluble complex of a proteolytic enzyme linked covalently to a polymeric substance such as polysaccharides.
U.S. Pat. No. 4,088,538 discloses a reversibly soluble enzymatically active polymer enzyme product comprising an enzyme covalently bonded to an organic polymer such as polyethylene glycol.
U.S. Pat. No. 4,415,665 discloses a method of conjugating an organic ligand containing at least one primary or secondary amino group, at least one thiol group and/or at least one aromatic hydroxyl group (described in col. 3, lines 19-36) to a polymeric carrier with at least one hydroxyl group (described in col. 2, lines 42-66).
U.S. Pat. No. 4,496,689 discloses a covalently attached complex of alpha-1-proteinase inhibitor with a polymer such as PEG or methoxypolyethylene glycols.
U.S. Pat. No. 3,619,371 discloses a polymeric matrix having a biologically active substance chemically bound thereto.
U.S. Pat. No. 3,788,948 discloses use of organic cyanate compounds to bind proteins to polymers.
U.S. Pat. No. 3,876,501 discloses activation of water-soluble carbohydrates with cyanogen bromide to improve their binding to enzymes and other proteins.
U.S. Pat. 4,055,635 discloses pharmaceutical compositions of a proteolytic enzyme linked covalently to a polymeric substance.
JP No. 5792435 published Nov. 26, 1982 discloses modified polypeptides where all or part of the amino groups are substituted with a polyethoxyl moiety. DE No. 2312615 published Sept. 27, 1973 discloses conjugating of polymers to compounds containing hydroxyl or amino groups.
EP No. 147,761 discloses a covalent conjugate of alpha-1-proteinase inhibitor and water-soluble polymer, where the polymer may be polyethylene glycol.
EP No. 154,316, published Sept. 11, 1985 to Takeda Chemical Industries, Ltd., discloses and claims chemically modified lymphokines such as IL-2 containing PEG bonded directly to at least one primary amino group of a lymphokine.
U.S. Pat. No. 4,414,147 describes rendering interferon less hydrophobic by conjugating it to an anhydride of a dicarboxylic acid such as poly(ethylene succinic anhydride).
Copending U.S. Ser. No. 749,955 filed June 26, 1985 discloses conjugation of PEG to such proteins as interferon-.beta., interleukin-2 and immunotoxins.
The heparin molecule consists of (1.fwdarw.4)-linked 2-amino-2-deoxy-.alpha.-D-glucopyranosyl, .alpha.-L-idopyranosyluronic acid and a relatively small amount of .beta.-D-glucopyranosyluronic acid residues.
Heparin has been bound covalently to solid supports to prepare blood-compatible surfaces. For example, PCT application WO86/03318 published Dec. 27, 1985 (Cardiol. Sci. Center) discloses immobilization of urokinase on a heparin support to produce a water-soluble complex with increased throbolytic activity. The conjugation is via a carboxyl group, not an aldehyde group.
It is desirable to conjugate the protein to a smaller heparin fragment. Hoffman et al., Carbohydrate Research, 117, 328-331 (1983) discloses a method for producing heparin fragments using nitrous acid, wherein the fragments have 2,5-anhydro-D-mannose residues as reducing terminal units with aldehyde groups. Such aldehyde groups may be reacted with primary amines to give labile Schiff-bases which can be converted to stable secondary amines by reductive amination. Hoffman et al. describes coupling of such heparin fragments to sepharose and curdlan and suggests coupling of heparin to human serum albumin and antithrombin.
None of the references, however, disclose how to use heparin fragments to water-solubilize recombinant proteins such as IL-2 and IFN-.beta. which are hydrophobic and therefore resist formulation in an aqueous medium at physiological pH. Furthermore, it is not a priori possible to predict which selected proteins would be favorably responsive, e.g., have good biological activity, to treatment with heparin fragments, due to the vast difference in physical properties between enzymes or albumins, on the one hand, and cytotoxins and lymphokines, on the other hand.